Osamu Yamasaki

Assessment of Streptococcus pyogenes microcolony formation in infected skin by confocal laser scanning microscopy

BACKGROUND Streptococcus pyogenes and Staphylococcus aureus are often simultaneously detected from many cases of non-bullous impetigo with atopic dermatitis. OBJECTIVES Using confocal laser scanning microscopy (CLSM), to investigate formation of S. pyogenes microcolonies in skin lesions. METHODS The S. pyogenes cells in the stationary growth phase alone were strongly stained with fluorescein isothiocyanate-concanavalin A (FITC-ConA), and this staining was reduced by pretreatment with amylase. Although the components of sugars in glycocalyx produced by S. pyogenes cells are unknown, we suggested that the materials stained by FITC-ConA were consistent with the presence of ConA-reactive sugars in glycocalyx produced by S. pyogenes cells. RESULTS S. pyogenes cells associated with streptococcal impetigo skin and croton-oil inflamed mouse skin formed microcolonies encircled by materials (glycocalyx) that stained strongly with FITC-ConA, and these findings were consistent with those in biofilms. In croton-oil inflamed mouse skin, polymorphonuclear leukocytes (PMNs) infiltrated to just below the epidermis in the cefdinir-treated group but only to the middle dermis in the cefdinir-non-treated group. In this case S. pyogenes and S. aureus cells formed separate microcolonies and existed independently in the outer walls of pustule lesions of streptococcal impetigo. CONCLUSION In skin infections, S. pyogenes and S. aureus formed aggregates of microcolonies (similar to that in biofilms) encircled by glycocalyx, which can make the infection hard to eradicate using an antimicrobial agent alone. The effect of conventional antimicrobial agents against biofilm is mainly due to the increase of the invasion of PMNs into the biofilm.

Effects of zinc oxide on the attachment of Staphylococcus aureus strains

We examined the attachment of Staphylococcus aureus to plastic tissue-culture coverslips after incubation for 24 h. The attachment to coverslips was weaker in rabbit plasma with 5% zinc oxide (ZnO) than in the control rabbit plasma without ZnO (P < 0.01). Plasma coagulation by S. aureus strains was not detected in plasma with 5% ZnO after incubation for 24 h. The membranous structure (an immature biofilm) was formed on the coverslips by S. aureus cells in plasma after incubation for 24 h. The colony counts of S. aureus cells on the membranous structures were lower in plasma with 5% ZnO, plasma with 0.2% hinokitiol, plasma with 5% ZnO + 0.2% hinokitiol, plasma with cefdinir at 4 minimum inhibitory concentration (MIC) and plasma with levofloxacin at 4 MIC, than in the control plasma after incubation for 24 h (P < 0.01). The colonies on the membranous structures completely disappeared in the case of plasma with 5% ZnO and 0.2% hinokitiol. The colony counts on membranous structures were lower in plasma with cefdinir at 4 MIC or levofloxacin at 4 MIC containing 5% ZnO than in plasma with cefdinir at 4 MIC or levofloxacin at 4 MIC only, (P < 0.05). The MICs of hinokitiol against S. aureus strains peaked at an MIC distribution of 16-32 micrograms/ml. The peak shifted to below 1 microgram/ml by adding 5% ZnO in agar plate method. The results suggest that the attachment of S. aureus cells to the coverslips is suppressed in the presence of 5% ZnO and that antistaphylococcal activities of cefdinir, levofloxacin and hinokitiol increase in the presence of 5% ZnO.

Confocal laser scanning microscopic observation of glycocalyx production by Staphylococcus aureus in mouse skin: does S. aureus generally produce a biofilm on damaged skin?

Summary Background Bacteria that adhere to damaged tissues encase themselves in a hydrated matrix of polysaccharides, forming a slimy layer known as a biofilm. This is the first report of detection of glycocalyx production by Staphylococcus aureus using confocal laser scanning microscopy (CLSM) on damaged skin tissues.

Confocal laser scanning microscopic observation of glycocalyx production by Staphylococcus aureus in skin lesions of bullous impetigo, atopic dermatitis and pemphigus foliaceus

Summary Background Glycocalyx collapses during dehydration to produce electron‐dense accretions. Confocal laser scanning microscopy (CLSM) may be used to visualize fully hydrated microbial biofilms.

Adherence characteristics and susceptibility to antimicrobial agents of Staphylococcus aureus strains isolated from skin infections and atopic dermatitis

We examined the adherence characteristics and susceptibility to various antimicrobial agents of 130 strains of Staphylococcus aureus isolated from infective skin lesions and 135 strains of S. aureus isolated from non-infective eczematous lesions of atopic dermatitis (AD) patients. The isolation rate of methicillin-resistant S. aureus (MRSA) was 27.7% in strains from clinical sources excluding AD and 31.1% in those from AD. Coagulase type II strains were most frequently observed in MRSA strains isolated from all sources excluding AD, and coagulase type III strains were most frequently observed in those isolated from AD. We proposed that antimicrobial treatment for AD patients should be carefully designed to prevent MRSA infection. Plasma coagulation ability was lowest in S. aureus strains isolated from abscesses, suggesting that the lower production of fibrin observed in abscesses may assist the infiltration of neutrophils into skin tissues and that a decrease in plasma coagulation ability may enable abscess formation. Adherence to polypropylene tubes with slime production was most evident in S. aureus strains isolated from felon and least evident in those isolated from cellulitis and lymphangitis. Tube adherence was characteristic of the S. aureus strains attached to superficial skin tissues, but not necessarily for strains that had infiltrated the deep skin tissues. Fusidic acid demonstrated significant antimicrobial activity against the MRSA strains, but rifampicin was the strongest antimicrobial agent.

Nasal and nasal-type natural killer/ T–cell lymphoma☆☆☆

Nasal and nasal-type natural killer (NK)/T-cell lymphomas follow an aggressive course and have a poor prognosis. Recent pathologic studies suggest that the disease is a malignant proliferation of NK cells, which often express CD56. An association with the Epstein-Barr virus has also been reported. Skin involvement occurred in each of the 3 patients studied. Radiation therapy provided some benefit to the patients in the early stages. Conventional chemotherapies were not effective. To overcome this multiple-drug resistance of the tumor cells, cyclosporine and high-dose chemotherapy was combined with peripheral-blood stem-cell transplantation. The average life span from the onset of the disease for our patients was 9.6 months. Further improvement in the management of nasal and nasal-type NK/T-cell lymphomas is necessary.

Induction of immune response against NY-ESO-1 by CHP-NY-ESO-1 vaccination and immune regulation in a melanoma patient.

BackgroundNY-ESO-1 is a cancer/testis antigen highly immunogenic in cancer patients. Cholesterol-bearing hydrophobized pullulan (CHP) is a nanoparticle-forming antigen-delivery vehicle and CHP complexed with NY-ESO-1 protein (CHP-NY-ESO-1) efficiently activates CD4 and CD8 T cells in vitro.AimIn this study we report on a 50-year-old male melanoma patient with multiple skin and organ metastases (T4N3M1c) who was vaccinated with CHP-NY-ESO-1 at biweekly intervals and who had an unusual disease course. We characterized in this patient humoral and cellular immune responses, immune regulatory cells, and cytokine profiles in the peripheral blood and at local tumor sites.ResultsTen days after the second CHP-NY-ESO-1 vaccination (day 25), blisters appeared on the skin at the metastatic lesions associated with inflammatory changes. A skin biopsy showed the presence of many NY-ESO-1-expressing apoptotic melanoma cells as determined by a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) test. However, the tumors continued to grow, and the patient died of pulmonary failure due to multiple metastases on day 48. Serum antibody responses were detected after the second CHP-NY-ESO-1 vaccination and antibody titer increased with subsequent vaccinations. Th1 dependent IgG1 was the predominant immunoglobulin subtype. Both, NY-ESO-1-specific CD4 and CD8 T cell responses were detected in PBMC by IFN-γ secretion assays. After CHP-NY-ESO-1 vaccination a slight decrease in CD4+CD25+Foxp3+ Tregs was observed in PBMC but significantly increased numbers of CD4+CD25+Foxp3+ Tregs and CD68+ immunoregulatory macrophages were detected at the local tumor sites. CD4+CD25+Foxp3+ Tregs were also increased in the blister fluid. Cytokines in the serum suggested a polarization towards a Th1 pattern in the PBMC and those in the blister fluid suggested a Th2-type response at the tumor site.ConclusionsOur observations indicate induction of specific humoral and cellular immune responses against NY-ESO-1 after CHP-NY-ESO-1 vaccination in a melanoma patient. The concomitant appearance of regulatory T cells and of immune regulatory macrophages and cytokines at the local tumor sites in this patient may explain immune escape.

Actions of Farnesol and Xylitol against Staphylococcus aureus

Background: Heavy colonization of atopic dermatitis (AD) with Staphylococcus aureus is well documented. The isolation rate of methicillin-resistant S. aureus is high in strains from AD in Japan. Our objective in the present study was to investigate the actions of farnesol and xylitol against S. aureus for the control of AD skin lesion-colonizing S. aureus.Methods: We examined the actions of farnesol on plasma coagulation and superantigenic exotoxin production by S. aureus, the antimicrobial activity of β-lactam antibiotics combined with farnesol at concentrations below the minimal inhibitory concentration (MIC) and the effect of xylitol on glycocalyx production. Results: Coagulation by S. aureus cells was inhibited in plasma containing farnesol at a concentration of 1/12 of the MIC (100 µg/ml) after incubation for 24 h. The production of superantigenic exotoxins by S. aureus cells with farnesol (100 µg/ml) was about 10 times lower than that by S. aureus cells alone. The MICs of ampicillin and cefdinir against S. aureus were reduced to ≤0.06 µg/ml in Mueller-Hinton agar plates with farnesol (100 µg/ml). We suggest that farnesol at concentrations above the MIC had a suppressive effect against S. aureus cells in the exponential and stationary phase and acted on the cell wall of S. aureus cells in both phases. Conclusions: Farnesol is a promising adjuvant agent against S. aureus skin infections treated with β-lactam antibiotics. Further, 5% xylitol inhibited glycocalyx production by S. aureus cells and consequently had a suppressive effect on the colonization of S. aureus on the horny cells of AD lesions.

Amicrobial pustular dermatosis in two patients with immunological abnormalities

Summary We report two patients with severe amicrobial pustular dermatosis with immunological abnormalities: a 63‐year‐old woman with a 30‐year‐history of discoid lupus erythematosus and sicca syndrome, and a 35‐year‐old woman with high levels of γ‐globulinemia and positive antinuclear antibodies. Both patients presented with crusty and eroded erythematous plaques studded with aseptic pustuleson the back, face, and scalp. Histological examination showed acanthosis, neutrophilic exocytosis to the epidermis, and neutrophilic and lymphocytic infiltration with nuclear dust in the dermis. These patients were diagnosed as having ‘amicrobial pustulosis associated with autoimmune diseases’. The eruptions improved with combination treatment of oral prednisolone with cyclosporin A or diaminodiphenylsulphone. Although the pathogenesis remains unclear, amicrobial pustular dermatosis might be one of the cutaneous complications in autoimmune diseases.

Confocal laser microscopic observation of glycocalyx production by Staphylococcus aureus in vitro

We used a scanning confocal laser microscope to study the effects of various agents on sugar production by Staphylococcus aureus in vitro. S. aureus cells attached to coverslips in Pl-TSB (plasma:tryptic soy broth=1:1) were stained with fluorescein isothiocyanate-conjugated concanavalin A (FITC-conA) and were more strongly stained over time. We considered that the materials that stained positive for FITC-conA consistent with S. aureus cells were sugars, probably glycocalyx, produced by the S. aureus cells. Since the cells in the stationary growth phase alone were strongly stained with FITC-conA, all S. aureus cells attached to the coverslips in Pl-TSB were considered to be in this phase (low growth rate). The positive staining for FITC-conA was markedly reduced when fibrin was not formed in Pl-TSB with plasmin and sucrose, and was also markedly reduced when the fibrin in Pl-TSB was destroyed with plasmin. In conclusion, the results of the present study indicate that the existence of fibrin is essential for glycocalyx production and biofilm formation of S. aureus cells to aid in the attachment of S. aureus cells in vitro, because S. aureus cells attached on coverslips and fibrin alone produce glycocalyx. Of the antimicrobial agents tested, sulfadiazine silver most strongly inhibited the production of FITC-conA-positive materials by S. aureus cells at a sub-MIC concentration. Plasmin, sucrose, and sulfadiazine silver may be useful topical applications for use on clinical dermatology for the prevention and the treatment of staphylococcal biofilms. We consider that this simple method is very useful for the detection of S. aureus glycocalyx on dermatology field.